1. Field of the Invention
The present invention relates to a process for preparing a therapeutic agent having the effect of restoring normal functions against dis-differentiated myelogenic leukemic cells from a culture liquid obtained by culturing a human amnion cell individually isolated by trypsinization treatment of human amnion membrane or a human amnion cell obtained by subculturing the isolated cell in a medium containing calf or fetal bovine serum at least one time, in a medium containing human serum albumin.
Myelogenic leukemia is a disease characterized by wide spread proliferation of the myleoid cells and the precursor cells in the bone marrow. The therapeutic agent prepared according to the process of the present invention acts against leukemic cells produced by the disorder of bone marrow to restore the normal function of the cells.
2. Description of the Prior Art
Medical research for methods of treating leukemia has hitherto been directed to the development of techniques which counter the cytotoxic effects of leukemia cell. However, recently the restoration of normal functions against dis-differentiated leukemic cells, that is, the development of a medicinal agent which promotes the decarcinogenesis of cancer cells has ben pursued (Chemistry and Biology, Vol. 13, No. 6, pp 380 to 382, 1975). Ichikawa et al. have described a process for preparing a medicinal agent which includes decarcinogenesis in which mammal fetal cells are cultured (Journal of Cellular Physiology, 74, 223, 1969; 76, 175, 1970 and Gann, 64, 257, 1973). Another conventional method is the Sachs et al method (Proceedings of the National Academy of Sciences of the United States of America, 70, 343 1973).
The Ichikawa et al method is as follows:
The fetus of a mouse is exenterated aseptically and sliced aseptically by a pair of scissors, and then a buffer solution containing trypsin is added thereto to isolate the fetal cells. The isolated cells are placed on a plate and a medium is added thereto in which the cells are cultured whereby primary culture cells are obtained. A suitable culture medium is the Eagle MEM medium combined with 10% (V/V) horse or calf serum. The cells grow in the form of a monolayer and are peeled from the glass surface of the plate with trypsin and cultured in the same medium again to obtain a secondary culture of the cells. In either case culturing is conducted in a carbon dioxide culture vessel containing 5% carbon dioxide at a temperature of 37.degree. C and a humidity of 100% for 3 to 5 days.
In the secondary culture liquid a substance which is capable of restoring normal functions against the dis-differentiated myelogenic leukemic cells of mouse to differentiated mature granulocytes or macrophages exists. The substance is known to be a non-dialysable high molecular protein which is inactivated by heating at 70.degree. C for 30 minutes or by treatment with a solution containing trypsin in a concentration of 0.4 mg/ml at 37.degree. C for 2 hours.
The Sachs et al. method is as follows:
The E1 cells which are established as a culture cell line from the fetus cells of a Swiss mouse are dispersed in an Eagle MEM medium combined with 10% of horse serum and placed in a plastic plate. After culturing the cells at 37.degree. C for 3 to 4 days whereby a monolayer of cells is formed, the monolayer is removed from the medium and is washed with Dulbecco's phosphate buffered saline (hereinafter referred to as PBS.sup..crclbar.) 3 to 4 times. Thereafter, an Eagle MEM medium not containing serum is added thereto and the cells are further cultured at 37.degree. C for 3 to 4 days. It is known that the culture liquid thus obtained contains a substance which is capable of restoring normal functions to dis-differentiated myelogenic leukemic cells of the mouse thereby differentiating to mature granulocytes or macrophages. The active substance is refined to an electrophoretically single protein by subjecting the culture liquid to ultra-filtration with a Diaflo membrane, Hydroxylapatite column chromatography, DEAE-Cellulose column chromatography and Sephadex G-150 column Chromatography in that order. It is known that this substance is a protein of about 68,000 in molecular weight and does not contain cystine, cystein, hexosamine and sugars and is inactivated by pronase treatment. However, these conventional methods have the following deficiencies:
As recognized by Dr. Ichikawa himself, the Ichikawa et al method has the deficiency that there is about four times the difference in the effect of a culture liquid for the restoration of normal functions against dis-differentiated leukemic cells depending upon the kinds of animal serum added to the growth medium and upon the lots of serum used even if the serum is derived from the same kind of animal. (Experimental Research, 90, 20, 1975) The Sachs et al. method is specific only to E1 cells established as a cultured cell line. It is apparent from further research on various cells established as a cultured cell line, that a substance which has the ability to restore normal functions to cells, as Dr. Sachs et al. insist is present, has not been identified as present in the culture liquid.
Recently, Dr. Maeda et al. have reported that a culture liquid containing a substance which has a normal restorative effect against dis-differentiated leukemic cells in a a high concentration is obtained by culturing animal fetus cells in a medium to which is added bovine serum albumin as a mammal serum in order to improve the conventional methods. (The 34th General Meeting of the Cancer Society of Japan, pp 127, 1975). Also, Dr. Ichikawa et al. have reported that a culture liquid having the same activity is obtained by culturing human fetus cells in a medium to which is added calf serum (Gann. 64, 3 247-263, 1973). However, these methods use cells of animals other than human beings or human fetus cells, sera or serum albumin of animals other than human beings. Therefore, when a medicinal agent derived from a culture liquid obtained by these methods is administered to a human being, an antigen-antibody reaction which originates from the serum or serum albumin of an animal other than a human being takes place so that the medicinal agent cannot be practically used. It is detrimental to public order or good morals to use human fetus cells. Thus, these conventional methods cannot be industrially employed.
As the result of investigating processes for preparing a therapeutic agent for myelogenic leukemia which does not induce an antigen-antibody reaction which is the most serious defect of the conventional methods, a culture liquid has been developed from cultured human amnion cells. These cells have never been used for preparing a therapeutic agent. The cells are easily available in media containing human serum albumin and have the effect of restoring normal functions to dis-differentiated myelogenic leukemic cells.